Fig. 3.

Targeting of endogenous mRNA by fluorescein-labeled antisense probes. (A,B) Detection of fluorescent puncta by different actb2 probes. One-cell-stage embryos were injected with 300 pg of indicated fluorescein-labeled probe and fixed at the four-cell stage for confocal microscopic imaging. Representative images are animal-pole views (A) with the cell border demarcated by a white-dashed line and magnification of the boxed area in the inset. The number of puncta (B) was calculated using NIS-element software under the same setting parameters. Each dot represents a single embryo. Ne, number of observed embryos. (C) actb2 probe-induced puncta are mainly dsRNA-positive. Embryos were injected at the one-cell stage with 300 pg fluorescein-labeled sense or antisense actb2 P3 probe and fixed at the four-cell stage for immunostaining with dsRNA antibody. (D,E) eomesa (D) or ybx1 (E) antisense probe-induced puncta in wild-type or MZ mutants. Top, relative position and length of in situ hybridization probes and fluorescein-labeled antisense probes to the target mRNA. Bottom, in situ hybridization pictures and fluorescent confocal images with magnification of the boxed area in the inset. Images show animal-pole views at the four-cell stage. (F) Number of fluorescent puncta formed in wild-type (WT) or MZeomesa or MZybx1 embryos. (G-I) Intensity (G) or number (H,I) of fluorescent puncta induced by indicated actb2 antisense probes. One-cell-stage embryos were injected with 300 pg of indicated single probes or probe mix (100 pg each) and observed at the four-cell stage. In B, F-I, each dot indicated one embryo; Ne, number of observed embryos. Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (Welch's t-test). ns, not significant. Scale bars: 100 µm (A,D,E); 10 µm (C).